The Lizard Log

The Langkilde Lab in Action


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A Frog of a Different Color: Sexually Dimorphic Color in Wood Frogs

Jett Peng is currently a senior majoring in Biology and minoring in German at Penn State. As an international student from Taipei, Taiwan, he has been embracing the American university life by learning all about American sports, food, and culture. He hopes to apply his laboratory experiences and knowledge to a pharmaceutical setting in the future. Jett describes his experiences in the lab below:

Last semester, I applied and received an Eberly College of Science Undergraduate Research Grant for my research project dealing with sexual dimorphism in wood frogs. Sexual dimorphism is a physical difference between males and females of the same species. These differences can include color, size, structure, shape, behavior, and much more.

DimorphEx

Two examples of sexual dimorphism: On the left, a male mallard (bottom) has a green head while a female mallard (top) has a brown head. On the right, the size difference between a female (left) and male (right) Argiope appensa. Photos from wikipedia.org

Humans, for example, are sexually dimorphic between males and females with differences in hair, height, and muscle mass. A fascinating thing about sexual dimorphism is the various functions it can have. Some sexual dimorphisms can be used as a defense mechanisms, such as warning signs or camouflage, or they can be used as breeding signals. Much research is being done into understanding what the exact functions of sexual dimorphisms can be and how they came about. One step into further understanding the origins of sexual dimorphism is to know when sexual dimorphisms first develop in an organism, which is what my experiment focused on.

The goal of my experiment was to determine if wood frogs (Lithobates sylvaticus) developed sexual dimorphism as juveniles. I worked with Lindsey Swierk and Brad Carlson, two graduate students in the Langkilde Lab, to help analyze data to see if juvenile frogs were sexually dimorphic. Previous research has shown that wood frogs are sexually dimorphic with female frogs being redder than males.

Male wood frogs are more brown (left) and female wood frogs are more red (right).

Male wood frogs are more brown (left) and female wood frogs are more red (right).

Knowing when the frogs begin to show this color change could give more information on the function of dichromatism; for example, it could serve as a function to prepare the wood frogs to become a perfect color for the breeding season.

To see if the juvenile wood frogs were sexually dichromatic, I analyzed photos of the frogs that were taken 1, 3, and 5 months after they underwent metamorphosis, the developmental process by which tadpoles change into frogs. The photos were analyzed and compared in the computer program, Photoshop, to obtain Red, Green, and Blue (RGB) values. The RGB values are of great importance for this experiment because those are the primary colors that are used to make an array of colors depending on the proportion of each color.  Color samples were analyzed from the back of the frogs because this was the area where the colors were most vibrant and noticeable.

Woodfrogcolorsample

Color sample was taken by using the circle shape tool in Photoshop to select the back area and analyze it for RGB values.

Finding the RGB values on the back of the frog allowed us to know the exact proportions of each color present; we would expect females to have a higher red value than males. The frogs were euthanized and then dissected to confirm the sex of each.

Preliminary results from the photographs (examples below) showed that wood frogs do display sexual dichromatism as juveniles and that they began to express sexual dichromatism about 3 months after metamorphosis.

FCompare

Photographs of a female wood frog taken from July (left), September (center), and November (right) of the juvenile stage.

MCompare

Photographs of a male wood frog taken from July (left), September (center), and November (right) of the juvenile stage.

Females tended to be redder and bigger than the males, with the males being more brown and smaller. These results are a basic foundation that can be further used to investigate why the frogs begin to experience color changes at around 3 months. Could the color changes be due to preparation for the breeding season or advantageous timing as a defense mechanism? What are the color differences between a juvenile and adult frog? These are some of the follow-up questions we can address to understand the development and function of sexual dichromatism in wood frogs.

Overall, this experiment was a very good experience for me where I learned many scientific research techniques. This individual research project allowed me to gain some experience in what it was like to do research. This project helped me become more organized as there was no set “due date” for the project, but I had to work at my own pace. I had to make sure I was doing all the work I needed to do for each week, so I could have enough data and time to work on a poster presentation for the Undergraduate Exhibition. The exhibition allowed me to communicate my scientific work in words for others to understand, which was very interesting. I really enjoyed doing this experiment because I learned how to obtain, record, and interpret results in a professional way. I am looking forward to help writing a manuscript next semester for this project.


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Lizards don’t do math.

Being stressed out is something everybody can relate to. Driving in heavy traffic, taking an exam, interviewing for a job, or dealing with family issues could all produce a similar stress response. Humans aren’t the only ones to experience stress. Zebras and lizards may not do math problems, but they may flee from or respond to a dangerous predator. These types of short-duration stressors are called “acute” and often involve only a single event. In humans, that might involve driving in heavy traffic or doing some difficult mental math. In the ecological world, acute stressors can include fighting with a competitor for food or territory or an encounter with a predator. Stressors that are persistent or long-lasting are called “chronic.” In humans, this could include getting over a recent break-up, being unemployed, caring for sick relatives, or worrying about money problems. In other animals, chronic stress could include exposure to a long winter storm or drought or continued difficulty finding food.

Life is stressful...

Life is stressful…

Although the kinds of stressors are quite different, the way an animal’s (or human’s) body responds is actually quite similar in most vertebrates. This stress response includes a suite of physiological changes that happen inside the body to help deal with the stressor. Part of what helps encourage these responses is the production of “stress hormones,” like cortisol in humans and fish or corticosterone in reptiles, amphibians, and birds. This suite of changes is often thought of in terms of energy:  stress hormones help redistribute energy toward things that are immediately important, like escaping from a predator. This energy is taken away from functions like reproduction or growth, which are not as important in the short-term.

The stress response is very important for dealing with stressors, but what happens when the stressor doesn’t stop or is frequently repeated for long time (e.g. is chronic)?  Under conditions of chronic stress, growth, reproduction, and immune function are often suppressed. Perhaps frequently taking energy away from these processes is not the best idea if it means an animal won’t be able to become big enough to mate or successfully produce offspring.

Lizards get stressed too.

Lizards get stressed too. (Photo by Gail McCormick)

If you’ve ever been in the Southern US, I’m sure you can relate to the stressful experience of being bitten by a fire ant! Fire ants can bite, sting, and even kill a lizard, so it makes sense that they have elevated levels of stress hormones (corticosterone) after a fire ant attack. Because they are frequently attacked, these lizards experience chronic stress, which in theory should lead to immune suppression. This got Tracy and I thinking:  although they have some neat ways of dealing with fire ants, lizards from fire ant invaded sites may frequently get bitten, which breaks the skin, or stung (envenomated), which activates the immune system. Thus, it seems that the immune system is important to lizards at invaded (high-stress) sites and that suppressing the immune system under chronic stress would be a bad idea for these lizards. Perhaps populations from invaded high-stress sites have adapted so that they do not suppress their immune function under chronic stress.

Female fence lizard. (Photo by Gail McCormick)

Lady fence lizard. (Photo by Gail McCormick)

Last year (summer 2012), we designed a project to determine if this were the case. I simulated the elevation of stress hormones that occurs from a fire ant attack by directly applying corticosterone (CORT) to the backs of lizards. (Corticosterone is dissolved in sesame oil, which is easily absorbed through the lizard’s skin.) We applied CORT every day for 23 days to lizards from invaded high-stress and uninvaded low-stress sites. Some lizards received just oil each day, to act as a control and base of comparison. At the end of treatments, we measured immune function to see if lizards from invaded and uninvaded sites responded differently to this chronic stress.

We expected all lizards that received CORT treatments would have reduced immune function compared to lizards that received just oil (control lizards). Additionally, we expected that this reduction would be less in lizards from invaded high-stress sites versus those from uninvaded low-strew sites. The graph below demonstrates this expectation. Taller bars indicate “better” immune function.

We expected all lizards to have reduced immune function following 2 days of CORT-treatments, but that this decrease wouldn't be so dramatic in lizards from high-stress sites.

We expected all lizards to have reduced immune function following 23 days of CORT treatments, but that this decrease would not be so dramatic in lizards from high-stress sites.

We used two different measures of immune function, both of which roughly measure the ability of lizard blood to deal with foreign particles, in this case E. coli (not a virulent strain!) and sheep red blood cells. Surprisingly, lizards that received CORT treatments actually had higher immune function compared to lizards that received just oil. Additionally, lizards from both invaded high-stress and uninvaded low-stress sites responded the same way to treatments.

What we actually saw: lizards from both high- and low-stress sites had enhanced immune function after CORT-treatments.  (idealized data for better presentation)

What we actually saw: lizards from both high- and low-stress sites had enhanced immune function after CORT-treatments.
(idealized data for better presentation)

There are may reasons why me may have seen these results. Here are a few of our many ideas:

  • Our lizards were fed a healthy diet of tasty crickets–likely more than they would eat in the wild. Perhaps for immune suppression to occur under chronic stress, lizards need to be food (and thus energy) limited. (With a healthy supply of energy, there may have been no need to divert energy from other systems like the immune system to deal with the stressor.)
  • Perhaps some parts of the immune system are more important than others. Some components that we did not measure in this study may be “okay” to compromise under stress in this context, while the components we did measure may not.
  • Perhaps CORT levels were able to return to “normal” between treatments, which makes this method more of a “repeated acute” stressor than a “chronic” stressor. We suspect that frequency and duration of a stressor are important predictors for consequences of stress, and I performed a study this past August to investigate this idea. I’ll be sure to share the results of that experiment in a future update!

These results were presented at the 2013 SICB annual meeting.

 

UPDATE: These results are published in General and Comparative Endocrinology: read it here!


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Backswimmers, Dragonflies, and Newts, Oh My!: How Large and Small Tadpoles Respond to Different Predators

This week in our undergraduate blogging, we’re featuring a post from Danielle Rosenberg, a senior in the Schreyer Honors College majoring in Veterinary and Biomedical Science and minoring in Equine Science. While at Penn State she has been the Community Service Chair in the Block and Bridle Club and  in charge of planning the largest blood drive on campus. She’s also just applied to veterinary school and  hopes to become an equine surgeon in the future

Last fall, I applied for and received an Eberly College of Science Undergraduate Research Grant to conduct an independent research project on wood frog tadpoles. Last spring, I planned my project with Brad, a graduate student in the Langkilde Lab, and waited for the ice to melt, the frogs to come out, lay their eggs, and for the eggs to hatch. I waited and waited and waited a little bit longer. The past spring happened to be a very cold one, and unfortunately the tadpoles hatched much later than expected which delayed my research plans, but did not deter me from addressing the following questions: “What risks do specific predators impose on tadpoles of various sizes, and how do tadpoles respond to these threats?

Tadpoles are very important components of aquatic ecosystems. They help to cycle nutrients through the ecosystem by feeding on detritus and on phytoplankton and periphyton, which are both primary producers in aquatic ecosystems and provide energy for living organisms. Tadpoles’ role in an ecosystem can be drastically affected by the presence of specific predators. Predators can have a huge effect on an ecosystem by not only eating the prey and decreasing their population density, but by also causing prey to react to the predator’s presence and change their behavior in some way. For example, tadpoles are known to reduce their activity in the presence of predator to avoid detection. Behavioral and morphological changes brought about by predator presence can cause the allocation of resources to change from reproduction to camouflage or defense mechanisms, greatly affecting a population and thus an entire ecosystem.

Tadpoles are prey for a number of insect and vertebrate predators, such as fish, newts, dragonfly larvae, salamanders, and water bugs. Each predator presents a unique level of risk to tadpoles based on their size, speed, effectiveness at capturing their specific prey, etc. and thus can cause varying responses on prey of different sizes. For example, smaller predators may be limited in the size of the prey they can eat (their mouthparts may only be so big!), while larger predators may not have that limitation. In addition, predators feed at different rates and eat varying numbers of tadpoles at a time. Similarly, the risks associated with each predator may change as tadpoles grow. As tadpoles age, they become larger and faster and may be able to escape or avoid some predators.

Wood frog tadpoles (Rana sylvatica) are a model system for this type of study because they are known to exhibit a strong response to predators and play an important role in aquatic ecosystems. For my study, I was looking to determine the susceptibility of tadpoles to various predators based on their size and to see if this influenced their behavioral response. To examine this I observed tadpole behaviors in response to a predator cue and followed-up with predation trials which allowed predators to feed on the tadpoles. I predicted that a more dangerous predator would cause a greater effect on the behavior of the tadpoles at their most susceptible size class.

Three of the different predators used; from R-L: a backswimmer, a dragonfly nymph, and an Eastern Newt

Three of the different predators used; from R-L: a backswimmer, a dragonfly nymph, and an Eastern Newt

The study seemed like it would be pretty straightforward and simple, but science never goes as expected! I started my research at the beginning of summer and had literally thousands of tadpoles at my disposal along with a group of predators that were known to consume tadpoles. I was using backswimmers, newts, dragonfly nymphs, and diving beetles as my predators and wood frog tadpoles as my prey. I would weigh tadpoles and separate them into two different size classes based on the limits I had set. One size class was 100 mg +/- 20%, and the other was 400 mg +/- 20%. I began weighing tadpoles and noticed some mortality of my populations in the lab, so Brad and I decided to move my research project out to the field where the environment might be less stressful on the tadpoles.

I began my research over again, weighing and sorting. Once I had sorted enough tadpoles into the two prescribed size classes, I began to run behavioral trials. For these I needed a predator cue to make the tadpoles think that a potentially dangerous predator was present during trials. I created this cue by allowing each predator type to eat a single tadpole in a small container and then collecting the water the predation event took place in. For the trials, I put 8 tadpoles from one of the size classes into 1800 mL of water and recorded their natural behaviors. I added 10 mL of regular water to the tubs as a control and observed the tadpoles’ movement every three minutes for a half hour and recorded the number of tadpoles swimming at each time interval. I then added 10 mL of the prescribed predator cue to each tub and again recorded the movements every three minutes for a half hour. The pre-predator cue data provided me with a way to detect tadpole behavioral changes due to the presence of the predator cue. I am still in the early stages of analyzing this data, but the preliminary analysis showed a decrease in tadpole activity across all predators and size classes. It did not seem to matter which predator or what size the tadpoles were; it seems as though the tadpoles respond by decreasing their behavior in the presence of any predator regardless of size. This makes sense as reducing activity might be a good way to avoid the attention (and subsequent attack) of potential predators.

An example of the two different size classes of wood frog tadpoles

An example of the two different size classes of wood frog tadpoles

Next, I ran predator trials to determine the susceptibility of each size class to the individual predators. I ran these trials by placing 8 tadpoles of a specific size class into a tub with a prescribed predator. I allowed each predator 2 hours to eat as many of the tadpoles as they desired. At the end of the 2 hour time period, the number of tadpoles remaining was recorded, and I am currently analyzing this data to determine predation rates on the two size classes.

These experiments will help to provide information that can be useful in understanding the workings of aquatic ecosystems and the various effects that predators can have on tadpoles during their various pre-metamorphic stages. Conducting this research was a great experience, and it gave me a new respect for the life of a researcher. While I always assumed that once a method was put in place, the experiment was just carried out, it turns out it does not always go exactly as planned. There are a lot of bumps and holes that need to be worked around and fixed to create a successful project. Some methods that look very simple on the outside probably went through a lot of revisions to get results and conclusions. Researchers need to have a lot of patience and be willing to constantly update and rework their methods. I think it is impressive how much time researchers can put into a project yet can seem so easy and laidback.


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Alabama Adventures

Hi, my name is Jennie. I’m an intern, on loan to the lab through Northeastern University’s co-op program. I spent March through May working at Penn State, assisting with a variety of projects in progress and conducting some independent research with Tracy’s guidance and support. I then tagged along on the lab’s second field excursion this past summer season, and I worked with a subset of the Langkilde cohort at the Solon Dixon Center in Alabama. Though much of my time was spent assisting with projects of the lab team , my personal research involved conducting experiments using Gastrophryne carolinensis, the Eastern Narrow-mouthed Toad, (which is actually an unusual type of frog, in the family Microhylidae).

JenniEGastro

An Eastern narrow-mouthed toad in all its glory

These little guys are fossorial, burrowing about pointy face first under logs and other cover. The summer is their reproductive season, when they can also be found in or around water by the males’ charming call – a high-pitched “waaaaaaaaaah” reminiscent of a sheep in distress or a cellphone vibrating on a countertop. They are ant specialists, with resilient, slimy skin and a protective turtle-neck like fold above the head to defend them from their prey. It has also been suggested that the gooey coating Gastrophryne secrete may contain defensive compound(s); one hypothesis suggests that these compounds originate from the venoms or toxins in the ants that they eat which may then be sequestered by the frogs.

G. carolinensis showing the distinctive skin fold on the neck which may protect them from attack by ants, their preferred prey

G. carolinensis showing the distinctive skin fold on the neck which may protect them from attack by ants, their preferred prey

G. carolinensis are a species of interest for the Langkilde Lab because their range overlaps with areas invaded by fire ants. In addition to displacing other species of ants which Gastrophryne typically eat, fire ants might impact the survival of individuals. It is clear that fire ants can damage Gastrophryne despite their specialized skin (they often have visible lesions after stings), but these frogs appear to be doing pretty well in invaded areas, as evidenced by their abundant calling, despite feeding on fire ants.

To examine whether the goo secreted by Gastrophryne might be an effective deterrent to fire ants, I designed the following experiment:

1)      Catch a bunch of Gastrophryne.  This necessitated extended, rapid log flipping in broiling heat and wading into flooded roadside ditches – 1 million thanks to Sean, Chris, and both Marks for their invaluable help!

2)      Put out yummy baits to attract fire ants, see previous post.

3)      Place a swab adjacent to the bait. I am comparing 4 types of swabs, rubbed thoroughly on one of the following: a Gastrophryne, a hotdog (which should attract the ants as a food source), some water (as a control) or a Hyla chrysoscelis, the Cope’s gray tree frog which definitely secretes something unpleasant (as do many amphibians; independently confirmed for these guys by the persistent burning sensation in my Smilax wounds after handling them).

GreyTreefrog

A Cope’s Grey Treefrog (Hyla chrysocelis). Photo credit: B. Carlson

JennieSwabbing

Me prepping for a fire ant trial by swabbing a narrowmouth toad.

4)      After 30 seconds, swiftly reclaim the swab and count the number of fire ants on it.

5)      After repeating 3) and 4) ad infinitum, collect the bait and pop it in the freezer to allowing counting of the foraging ants, so variation caused by the number of ants present can be accounted for.

I’m starting to process the data from this experiment, but I expect that: 1) the swabs with hotdog residue should have the most fire ants on them, and 2) if the secretions of the narrowmouth toads and treefrogs are indeed noxious to fire ants, that those swabs should have the fewest fire ants on them. We’ll see how the data analysis works out!

As is generally the case when implementing a new experimental procedure, I shortly encountered an unforeseen complication – Gastrophryne get pretty dried out after being swabbed multiple mornings in a row.  Additionally, when given time to regenerate their goo in captivity on a diet of termites, they preliminarily appear to be more attractive to the ants. Interesting! Maybe in future experiments we can examine how diet influences the noxiousness of Gastrophryne goo!

A second experiment I attempted was to evaluate feeding preferences of Gastrophryne for different species of native ants and fire ants collected around the Dixon Center. I was hoping the Gastrophryne would actively hunt when presented with a bevy of ants, so I could see if they preferred a particular species.  However, when placed in their novel Gladware environments they seemed too distracted to do anything but attempt escape or tuck into a corner…Poor little buddies! I got to see them zap any ants that blundered into their vicinity though; that was pretty neat.

On this summer’s trip, I was able to do all kinds of cool field biology, have a lot of fun, and learn constantly (being beleagueringly uninformed compared to my herpetologist compatriots, whose patience with my inexperience is greatly appreciated!) – Thanks to the Langkilde Lab for providing me with such an awesome opportunity!


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Washing Leaves and Spiking Feces: How to Trace Nutrients Through an Ecosystem

Continuing in our series of posts by Langkilde Lab undergraduates, we have a dispatch from Tyler Jacobs, a senior majoring in Immunology & Infectious Disease. Tyler has played the mellophone in the Blue Band as well as being in the Phi Mu Alpha Sinfonia Music Fraternity for all of his 4 years at PSU. After graduation he’ll be entering the Navy, getting a bachelors degree in nursing, and aiming to become a nurse anesthetist. Tyler’s post is below:

For the past year, my research has been focused on testing the accuracy and reliability of a new technique for field ecologists. The goal of my project is to determine whether nonradioactive (stable) isotopes can be used to trace the flow of nutrients through an ecosystem (specifically through the mangrove ecosystem).  Both macro- and micronutrients are important to the growth and development of plant tissues. This method can prove to be a useful new technique in future studies of nutrient cycles, such as the Nitrogen cycle, or, as in this case, and attempt to examine how an invasive species can affect nutrient flows within a specific ecosystem. In some mangrove ecosystems in Puerto Rico, invasive iguanas can cause severe damage by literally defoliating entire mangrove trees. These iguanas then do what all animals do after eating: they poop. This iguana gluttony on mangrove leaves can result in an accelerated delivery of nutrients, such as nitrogen, in the form of poop to other parts of the ecosystem. I attempted to describe this flow by using stable isotopes.

There were two components of this project: leaf assimilation of isotope and isotope uptake by animal tissues. The leaf assimilation involved the spraying of 15N isotope on three types of mangrove leaves in Jobos Bay, Puerto Rico. 15N, or “Nitrogen-15”, is an isotope of the naturally occurring element Nitrogen. However, while 15N is found naturally, it only makes up ~1% of the total nitrogen available to all living systems; most Nitrogen is 14N. The presence of an additional neutron (hence the number 15), can serve as a tag in various tests, allowing the nutrient to potentially be traced as it moves within an ecosystem. This type of research is often referred to as stable isotope research.

Red Mangrove Tree (Rhizophora mangle)

Red Mangrove Tree (Rhizophora mangle)

In our case, the sprayed leaves underwent a series of tests designed to strip away the surface of the leaf (via simulated rain, or by rubbing soap on the leaf’s surface). The theory behind this madness was simple: if the leaf did not absorb the sprayed-on isotope, it was just sitting on the surface of the leaf within all of the oils that make up the “waxy cuticle” outer layer (which is also responsible for the shiny appearance of certain leaves). To prove that this isotope wasn’t just sitting in this oil layer, the soap was used to completely remove it, leaving behind nothing but the organic leaf tissue. Therefore, if we did find any isotope, we could be sure that it had indeed been absorbed into the tissue of the leaf. All of the samples were processed in the lab and analyzed by GCMS for their isotope composition. GCMS stands for gas chromatography-mass spectrometry. It is a commonly used method for accurately determining the nutrient concentrations of small organic samples.

The uptake of isotope by animal tissues was the second portion of the project. This experiment allowed us to discover whether animals can absorb isotopes that have already been processed and used by another organism. To put it simply, the main question we are asking is: if tree leaves are enriched with isotope (15N) and eaten by animals, will the animals process and retain the original isotope in their digestive and muscular tissue? We tested this theory by setting up 5 plots, each containing feces of I. iguana. These invasive iguanas had been fed with the 15N enriched leaves, producing poop spiked with 15N. We allowed the local bottom-feeders, or detritivores (coffee been snails and fiddler crabs) to roam freely among the plots over a weeklong time period, during which they ate the iguana feces (delicious!) along with other decaying matter. To determine whether these detritivores were taking up the 15N, each day, a single crab and snail were collected from each plot. In the laboratory, I removed the digestive organ of the crabs, known as the hepatopancreas. Both digestive (digestive gland) and muscular tissue (muscular foot) were removed from the snail samples. All of the animal samples were analyzed by GCMS to determine their isotope composition, similar to the leaf samples.

Fiddler crab (Uca rapax) taken in Jobos Bay, Puerto Rico

Fiddler crab (Uca rapax) taken in Jobos Bay, Puerto Rico

The results from the isotope analysis of the leaf assimilation portion of the project showed promising results. All of the mangrove leaf samples retained a large amount of 15N in comparison to the control leaves. This result demonstrates that the method of spraying isotopes on leaves is an effective means of monitoring nutrient flow. The results of the animal tissue portion of the experiment proved generally inconclusive. We saw virtually no differences between the control concentration of 15N and the tissues collected over the weeklong period. This suggests that, while mangrove leaves took up the 15N, the isotope did not flow through the food web all the way to the detritivores (or at least was not at detectable levels).

To sum it all up, our main goal was to prove that this particular method of tracing nutrients through an ecosystem works. We discovered that although the animals did not take up the isotope as anticipated, the leaves were able to absorb 15N into biological tissues quite effectively. Although not as effective of a method as we had initially planned, this use of stable isotopes still proved useful in the tracing of nutrients through mangrove ecosystems.